The overarching objective of this work is to contribute to our fundamental understanding of how high fidelity in protein synthesis is achieved during translation of the genetic code. Accurate substrate recognition and discrimination by aminoacyl-tRNA synthetases (aaRS) is a key feature of this process. There are two classes of aaRS, and research in the Musier-Forsyth lab for the past sixteen years has revolved around class II synthetases, with a strong focus on prolyl-tRNA synthetase (ProRS). During the previous grant period, we began turning our focus to quality control mechanisms that prevent mistranslation due to the misactivation of structurally related amino acids by aaRSs. After misactivation, some noncognate amino acids are immediately hydrolyzed in the aminoacylation active site (i.e., the first or coarse sieve) in a reaction known as pre- transfer editing; others are attached to tRNA and translocated to an editing site that has been designated the second or fine sieve. The latter domain carries out a reaction referred to as post-transfer editing. ProRSs display an unusually diverse range of domain architectures. Bacterial enzymes contain an editing domain (INS) that is responsible for clearing misacylated Ala-tRNAPro. The INS domain is missing from most archaeal and eukaryotic enzymes, however, genome-encoded single-domain proteins (e.g., YbaK family) with homology to the ProRS INS domain are widely distributed in all three kingdoms. Interestingly, some members of this family have been shown to possess Cys-tRNAPro editing activity and to interact with the ProRS/tRNA complex. These observations allowed us to propose a novel triple-sieve mechanism of editing, which forms the basis of the present renewal application. Here, we propose to continue to explore the specificities and novel functions of these intriguing and poorly understood single-domain proofreading proteins, as well as their mechanisms and species-specific differences. The specific aims are: 1) To explore pre-transfer editing by ProRS, 2) to explore the post-transfer editing mechanism of ProRS and ProRS-like editing domains, 3) to explore the function of YbaK and homologous families of proteins in vitro and in vivo, and 4) to probe the structure of the ProRS/YbaK/tRNA ternary complex. PUBLIC HEALTH RELEVANCE: The objective of this work is to contribute to our fundamental understanding of how high fidelity in protein synthesis is achieved during translation of the genetic code. The aminoacyl-tRNA synthetases and related genome-encoded single domain proteins studied in this work, possess editing functions that prevent errors in protein synthesis. Editing-defective synthetases have been implicated in progressive neurodegenerative diseases. Thus, in recent years, it has become clear that a better understanding of the editing functions of synthetases may provide insights and novel treatments for various diseases. Due to their essential function, these enzymes are also promising targets for the development of antibiotics and antifungal agents.